The use of ethanol as an automotive fuel provides a cleaner burning and renewable alternative to petroleum-based fuels [1]. Technology currently in use for ethanol production is based on edible crops such as sugar cane juice (molasses) and corn starch [2] that have alternative markets. The cost of these feedstocks has been estimated to represent 40% of total production costs [3]. In contrast, inedible lignocellulosic biomass is available at a cost competitive with petroleum [4]. The continued development of improved microorganisms for the conversion of lignocellulosic sugars into ethanol offers the potential to decrease dependence on petroleum and create new manufacturing opportunities from existing plant materials.
Ethanologenic strains of Klebsiella oxytoca have been developed [5, 6]. These strains have been shown to metabolize a variety of sugar monomers (such as glucose, xylose, and arabinose) derived from lignocellulosic biomass [5-7]. Such strains can function well in simultaneous saccharification and fermentation (SSF) processes with cellulose [8-10]. An ethanologenic strain of K. oxytoca known as K. oxytoca P2 has been described that contains genes from Zymomonas mobilis encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB), enzymes involved in converting oligosaccharides to ethanol. These genes are chromosomally integrated into the genome of strain P2.
In contrast to analogous strains of Escherichia coli [11-13], K. oxytoca has the native ability to metabolize many soluble products from lignocellulosic biomass, including cellobiose, cellotriose, xylobiose, xylotriose, and arabinosides [6, 14, 15]. The ability of K. oxytoca P2 to efficiently metabolize incompletely hydrolyzed products from lignocellulose at pH 5.2 (near optimal for fungal enzymes) provides an added advantage during simultaneous saccharification and fermentation (SSF) processes [8]. Under these conditions, K. oxytoca P2 required less than half of the fungal cellulase required by Saccharomyces cerevisiae to achieve equivalent fermentation rates and yields [9].
The availability of inexpensive industrial media for growth of ethanologenic bacteria that support high ethanol productivity and yield is essential for ethanol production from biomass feedstocks [17, 18]. However, unlike grain, hydrolysates of cellulosic biomass are inherently nutrient poor and must be supplemented [16]. Accordingly, previous use of K. oxytoca P2 for ethanol production has involved culture of the cells in complex growth media containing laboratory nutrients such as yeast extract and Difco Tryptone™. Unfortunately, it is impractical to use such nutrients for commercial production of commodity chemicals such as ethanol from lignocellulose.
To fully realize the potential of recombinant ethanologenic bacterial strains to serve as a source of ethanol, there is a clear need for new and improved strains of such bacteria that can efficiently produce ethanol while growing in inexpensive minimal media, and new media that can support these cells.